1. Field of the Invention
The invention relates generally to methods of using nanoparticles for biomolecule analysis, and more specifically to methods of using a composite organic-inorganic nanoparticle (COIN) for detecting and quantitating a molecule by surface-enhanced Raman spectroscopy (SERS), and to methods of using a COIN to image a cell, including one or more molecules in a cell, by SERS.
2. Background Information
Multiplex reactions are parallel processes that exist-naturally in the physical and biological worlds. When this principle is applied to increase efficiencies of biochemical or clinical analyses, the principal challenge is to develop a probe identification system that has distinguishable components for an individual probe in a large probe set. High density DNA chips and microarrays are probe identification systems in which physical positions on a solid surface are used to identify nucleic acid or protein probes. The method of using striped metal bars as nanocodes for probe identification in multiplex assays is based on images of the metal physical structures. Quantum dots are particle-size-dependent fluorescent emitting complexes. Quantum dots, which emit at short wavelength in response to UV excitation, are used for highly multiplexed detection.
Biochips, including DNA arrays (DNA chips), microarrays, protein arrays and the like are devices that may be used to perform highly parallel biochemical reactions. Such devices are fabricated either by building the biomolecules (nucleic acids or proteins) as probes on the chip surface directly or depositing the biomolecules on the chip surface after they have been synthesized. Generally physical positions (XY coordinates) are used to identify the properties or sequences of detected probes molecules.
Conventional cell imaging techniques have used fluorescent dyes. Current research (for example, Jaiswal et al. (2003) Nature Biotechnology 12:47-51) has turned to use of quantum dot technology for cell imaging because of their extended lifetime. Also emission spectra from quantum dots are narrower, allowing more colors to be used together. Traditional methods for protein profiling have utilized, for example, two-dimensional gel and MDLC-MS (Multi-dimensional Liquid Chromatographs-Mass Spectroscopy).
The ability to detect and identify trace quantities of analytes has become increasingly important in virtually every scientific discipline, ranging from part per billion analyses of pollutants in sub-surface water to analysis of cancer treatment drugs in blood serum. Raman spectroscopy is one analytical technique that provides rich optical-spectral information, and surface-enhanced Raman spectroscopy (SERS) has proven to be one of the most sensitive methods for performing quantitative and qualitative analyses. A Raman spectrum, similar to an infrared spectrum, consists of a wavelength distribution of bands corresponding to molecular vibrations specific to the sample being analyzed (the analyte). In the practice of Raman spectroscopy, the beam from a light source, generally a laser, is focused upon the sample to thereby generate inelastically scattered radiation, which is optically collected and directed into a wavelength-selective spectrometer in which a detector converts the energy of impinging photons to electrical signal intensity.
Among many analytical techniques that may be used for chemical structure analysis, Raman spectroscopy is attractive for its capability in providing rich structure information from a small optically focused area or detection cavity. Compared to a fluorescent spectrum that normally has a single peak with half peak width of tens of nanometers (quantum dots) to hundreds of nanometers (fluorescent dyes), a Raman spectrum has multiple bonding-structure-related peaks with half peak width of as small as a one nanometer, or less. Surface enhanced Raman scattering (SERS) techniques make it possible to obtain a 106 to 1014 fold Raman signal enhancement, and may allow for single molecule detection sensitivity. Such huge enhancement factors are attributed primarily to enhanced electromagnetic fields on curved surfaces of coinage metals. Although the electromagnetic enhancement (EME) has been shown to be related to the roughness of metal surfaces or particle size when individual metal colloids are used, SERS is most effectively detected from aggregated colloids. Chemical enhancement may also be obtained by placing molecules in a close proximity to the surface in certain orientations. Due to the rich spectral information and sensitivity, Raman signatures have been used as probe identifiers to detect a few attomoles of molecules when SERS method was used to boost the signals of specifically immobilized Raman label molecules, which in fact are the direct analytes of the SERS reaction. The method of attaching metal particles to Raman-label-coated metal particles to obtain SERS-active complexes has also been studied. A SERS signal also may be generated after attaching thiol-containing dyes to gold particles followed silica coating.
Analyses for numerous chemicals and biochemicals by SERS have been demonstrated using: (1) activated electrodes in electrolytic cells; (2) activated silver and gold colloid reagents; and (3) activated silver and gold substrates. However, none of the foregoing techniques is well suited for providing quantitative measurements. Consequently, SERS has not gained widespread use. In addition, many biomolecules such as proteins and nucleic acids do not have unique Raman signatures because these types of molecules are generally composed of a limited number of common monomers.
SERS technique has become an important analytical tool because it may identify and detect single molecules without labeling. SERS effect is attributed mainly to electromagnetic field enhancement and chemical enhancement. It has been reported that silver particle sizes within the range of 50-100 nm are most effective for SERS. Theoretical and experimental studies also reveal that metal particle junctions are the sites for efficient SERS.